The high prevalence of obesity in the US is a major public health concern, as overweight and obese individuals are at increased risk for many chronic diseases. Obesity stems from an imbalance between total caloric consumption and total energy expenditure (TEE), therefore accurate measurements of TEE play a pivotal role in understanding and ultimately reversing this epidemic. The gold standard for measuring TEE in free-living individuals is the doubly labeled water (DLW) method. The major limitations of the DLW method include high cost and precision of only 1 5% for individual measurements. In this Small Business Innovative Research (SBIR) project, Los Gatos Research (LGR) will develop, fabricate, and test a novel, laser-based analyzer to dramatically improve DLW measurements of TEE by concurrently measuring 17O/16O to correct for natural isotopic fluctuations in 18O and 2H. The Triple Isotope Water Analyzer (TIWA), which will be based on LGR's patented, laser-based Off-Axis ICOS technology, will be capable of real-time, simultaneous, high-throughput (50 samples/day) monitoring of 2H/1H, 18O/16O, and 17O/16O in H2O from human bodily fluids (e.g., blood, urine, and plasma), with a target accuracy of better than 10.60 for 2H/1H, 10.20 for 18O/16O, and 10.30 for 17O/16O at natural isotopic abundances. For enriched body waters that have been labeled with 2H and 18O, the target accuracy will be comparable to isotope ratio mass spectrometry and better than 11.50 for 2H/1H, 10.50 for 18O/16O. The TIWA will allow quantification of isotopic background fluctuation during measurements of TEE, eliminating the significant "constant background" assumption made during DLW experiments. Measurement of the isotopic background fluctuation will allow for much smaller isotopic doses to be used, substantially reducing the cost of TEE measurements. Alternatively, these measurements can be used to significantly increase the accuracy of the DLW technique, introducing the possibility of extending this technique from population studies to the accurate assessment of the TEE of individual subjects. During Phase I, LGR will work with Professor Edward Melanson, an established researcher of metabolic studies, and Professor John Speakman, a key developer of the DLW method, to demonstrate a prototype instrument for measurements using vacuum-distilled, clinical samples. The correlation in background fluctuations between 2H/1H, 18O/16O, and 17O/16O from 40 humans will be quantified to demonstrate the applicability of using the 17O/16O signal to correct for background fluctuations in DLW experiments. Finally, two individuals will be measured using the DLW method to directly compare the conventional double isotope measurement to the newly-developed triple isotope measurement. At the conclusion of this research project, LGR will have demonstrated the use of the TIWA for DLW measurements and empirically determined the improvement in TEE accuracy and reduction in the DLW method cost due to the measurement of background fluctuation using 17O/16O. PUBLIC HEALTH RELEVANCE: The high prevalence of obesity in the US is a major public health concern, as overweight and obese individuals are at increased risk for many chronic diseases. Obesity stems from an imbalance between total caloric consumption and total energy expenditure, therefore accurate measurements of total energy expenditure (TEE) play a pivotal role in understanding and ultimately reversing this epidemic. This Small Business Innovative Research (SBIR) project will allow direct quantification of isotopic background fluctuation during doubly-labeled water measurements of TEE, potentially reducing the cost of isotope dose for TEE measurements from approximately $400 to $80 per adult subject and thereby greatly increasing the application of TEE measurements for obesity studies.